Approximately 90% of all cancer deaths arise from the metastatic spread of primary tumours. Of all the processes involved in carcinogenesis, local invasion and the formation of metastases are clinically the most relevant, but they are the least well understood at the molecular level. As a barrier to metastasis, cells normally undergo an apoptotic process known as 'anoikis', in circulation. The recent technological advances in the isolation and characterisation of rare circulating tumour cells (CTCs) will allow a better understanding of anoikis resistance. Detailed molecular and functional analyses of anoikis-resistant cells may provide insight into the biology of cancer metastasis and help identify novel targets for prevention of cancer dissemination. To uncover the molecular changes that govern the transition from a primary lung tumour to a secondary metastasis and specifically the mechanisms by which CTCs survive in circulation, we carried out whole genome sequencing (WGS) of normal lung, primary tumours and the corresponding brain metastases from five patients with progressive metastatic non-small-cell lung carcinoma. We also isolated CTCs from patients with metastatic cancer and subjected them to whole genome amplification and Sanger sequencing of genes of interest. While the primary tumours showed mutations in genes associated with cell adhesion and motility, brain metastases acquired mutations in adaptive, cytoprotective genes involved in response to cellular stress such as Keap-1, Nrf2 and P300, which are key players of the Keap1-Nrf2-ARE survival pathway. Nrf2 is a transcriptional factor that upon stress translocates into the nucleus, binds to the anti-oxidant response elements (ARE) and drives the expression of anti-oxidant genes. The identified mutations affect regulatory domains in all three proteins, suggesting a functional role in providing a survival advantage to CTCs in the peripheral blood allowing their dissemination to distant organs.

Glioblastoma Multiforme (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. The majority of GBM tumors show a mutated or overexpressed Epidermal Growth Factor Receptor EGFR; however, tumors treated with EGFR inhibitors such as gefitinib or erlotinib inevitably recur. This recurrence highlights the need to identify signaling pathways involved in GBM resistance to EGFR therapy that may serve as targets for intervention. In our recently published work, we found that activation of the ROS1 pathway is a primary mechanism by which glioma cells become resistant to EGFR‐targeted therapy. ROS1 is a proto‐oncogene receptor tyrosine kinase activated by chromosomal rearrangement in several human cancers, including non‐small‐cell lung cancer (NSCLC), cholangiocarcinoma, gastric cancer, ovarian cancer, and glioblastoma. We therefore, designed, synthesized and screened a series of new ROS1 inhibitors. The lead compound, ROS‐Ic, is very potent (IC50 = 24nM) and highly specific to ROS1 protein. Treatment of gefitinib‐resistant GBM cells with ROS1‐lc sensitized the resistant cells to low concentrations of gefitinib resulting in apoptosis following a prolonged S phase cell cycle arrest. When compared to Crizotinib, an FDA‐approved small‐molecule tyrosine kinase inhibitor currently used to treat ALK‐ and ROS1+ NSCLC patients, ROS‐Ic showed superior cytotoxic activity in vitro towards ROS1‐positive GBM cells. Crizotinib resistance is thought to be due to diminished Crizotinib binding, due to ROS1 mutations in Crizotinib binding pocket. Interestingly, our initial analysis suggests that our lead compound ROS1‐Ic might not be affected by these mutations, highlighting the potential therapeutic value of this compound. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.